Immune system gene polymorphisms associated with severe dengue in Latin America: a systematic review

ABSTRACT One of the main challenges in the clinical management of dengue is the early identification of cases that could progress to severe forms of the disease. A biomarker that may enable this identification is the presence of genetic polymorphisms in genes associated with immune responses. The objective of this study was to perform a systematic review of the Latin American literature on these genes. An electronic literature search was carried out in PubMed, Scopus, Lilacs, and the Virtual Health Library, and reference lists of systematic reviews in the area. Case-control studies conducted in Latin American countries examining at least one form of genetic polymorphism related to immune responses against severe dengue were included. In total, 424 articles were identified and 26 were included in this systematic review. Of the 26 selected articles, 16 reported polymorphisms associated with the risk of developing severe dengue (Risk); Similarly, 16 articles reported polymorphisms associated with a decreased risk of severe dengue (Protective). The final analysis revealed that multiple polymorphisms in immune system genes were early markers of the progression of dengue in Latin Americans and found that polymorphisms of the TNF-alpha gene may have a critical role in dengue pathogenesis.


BACKGROUND
The bite of Aedes mosquitoes, which can be found in more than 100 countries, transmits an RNA-type virus that causes acute dengue and belongs to the Flaviviridae family 1 .It is estimated that about 3 billion people live in areas with increased dengue risk 2 .While around 390 million new cases are reported yearly, roughly 75% are asymptomatic and not included in each nation's official statistics 3 .This means that about 96 million dengue infection cases occur annually, with symptoms requiring health care attention 4 .In the Americas, 1,173,674 dengue cases were reported in 2021, of which 2,821 (0.24%) were severe dengue cases 5 .
The following factors have been found to increase the severity of the clinical manifestations of dengue: Antibody Dependent Enhancement (ADE) 6 ; the immune response mediated by the dengue virus (DENV) serotype that first infects a patient (DENV1, DENV2, DENV3, DENV4) and the order of subsequent infections [7][8][9][10] ; age at the moment of the disease [11][12][13][14] ; pre-existing co-morbidities (especially diabetes and renal disease); and the presence of warning signs.Additionally, the clinical signs of a secondary dengue infection are often minor when it occurs less than two years after the first 15,16 ; however, intervals between two infections greater than four years have been linked to more severe clinical manifestations 17 .
The presence of allelic variants in the coding sequences for the major histocompatibility complex type B (MIC-B) and for phosphoinositide phospholipase C epsilon 1 (PLCE1) 18 , as well as African ancestry 19,20 , are findings that link the individual's genetic profile with the severity of dengue in individuals.Other genes control the inflammatory response, such as CD209, which produces the DC-SIGN dendritic cell receptor, and the tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine involved in the regulation of immune responses, cell proliferation, differentiation, and apoptosis.These genes also seem to have an essential role in controlling individuals' susceptibility to severe dengue.Additional examples of genes involved in the control of dengue are the FcRIIA, which codes for Fc-type receptors expressed in antigen-presenting cells and is directly involved in the mechanisms of antibodydependent enhancement (ADE) 21 , and genes that encode for toll-like receptors (TLRs), which are involved in the activation of innate immunity cells.
No specific treatment for dengue has been established to date, and the development of prophylactic vaccines is still incipient in managing the disease 22 .In fact, incidences of dengue continue to increase, and the virus that causes it is still widely spread 23 .In this scenario, it is essential to continue exploring mechanisms that allow the early identification of severe cases, to improve clinical approaches and directly reduce the mortality rates of the disease.Therefore, we conducted a systematic review of the current literature to identify the genetic variants linked to the emergence of severe dengue (dengue hemorrhagic fever and dengue shock) in Latin American populations.

MATERIALS AND METHODS
This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 24 (Supplementary Table S1).The protocol was not registered before this review.

Search strategy
An electronic literature search was conducted in PubMed, Scopus, Lilacs, and the Virtual Health Library (VHL -BVS in Spanish).VHL is a specific database for the Americas.A search of the reference lists of systematic reviews in the area was also conducted [25][26][27][28] , combining MeSH and DeCS descriptors on dengue, polymorphism and the countries of Latin America.Specific terms were used to search the four databases, emphasizing the search for titles and abstracts.The supplementary material contains the strategy applied to search each database (Supplementary Table S2).English and Spanish terms were combined:

Inclusion and exclusion criteria
Case-control studies conducted in a Latin American country that examined at least one genetic polymorphism related to immune responses to severe dengue were included.Our review only included studies that evaluated polymorphism with well-documented genetic tests: polymorphism detection with PCR-SSP (polymerase chain reaction-sequence specific of primers); RFLP (Restriction Fragment Length Polymorphism) for the restriction enzyme; qPCR (real-time PCR), and the Amplification-refractory mutation system (ARMS-PCR).Articles published up to November 3 rd , 2022, were included in the review.Studies on non-human genetics (e.g., viral genetics, mosquito genetics), case reports, letters to the editor, and other nonobservational studies were excluded.Studies that did not employ confirmatory tests for dengue were also excluded.Tests that adhered to the World Health Organization (WHO) guidelines 29,30 , using methods such as viral isolation techniques, detection of antigens or antibodies, and nucleic acid detection, were considered appropriate.Lastly, studies using laboratory-confirmed IgM ELISA or IgG ELISA and reverse transcription polymerase chain reaction (RT-PCR) methods were also included.

Study selection
Two authors (JESF and LSGC) independently reviewed titles and abstracts and identified potentially relevant articles, resolving discrepancies through further review and mutual consensus.Both investigators fully read all potentially relevant articles and determined the final reports to be included in this review.The data sets were extracted and organized in bibliographic tables.The primary fields contained information on the authors, country, year of publication, sample size for each study group, study objective, molecular test to determine the genetic polymorphism, the polymorphism evaluated, the group and type of immunity and WHO classification of the dengue cases (guideline 1997 or 2009).Types of immunity were classified according to Immunity Groups, which were based on the contributions of Harapan et al. 3 , and Bhat et al. 31 : Group A) Innate immunity; Group A1) Interferons and interleukins; Group A2) Mannose-binding lectin (MLB2); Group A3) Others (histidine, serotonin, complement and nitric oxide); and Group B) Genetic (HLA).

Quality assessment
The quality of each study was assessed using the Newcastle-Ottawa Quality Assessment Scale for Case-Control Studies (NOS) 32 , which evaluates the selection, comparability, and exposure determination of each study, and independently assessed by two authors (JESF and LSGC).Based on the NOS score and quality classification, the risk of bias in the initially selected studies was evaluated and classified as high risk (NOS≤6), some concerns (NOS=7), and low risk (NOS≥8).The study quality evaluation was summarized in a figure specifying whether studies met the criteria (green: low risk of bias), did not meet the criteria (red: high risk of bias), or if results were unclear (yellow: unclear risk/some concerns/lack).Supplementary Table S3 shows the scoring criteria based on the Newcastle scale.

Search results and article selection
In total, 424 bibliographic references were found: 97 in PubMed, 108 in Scopus, 20 in Lilacs, and 190 in VHL.Nine articles were obtained during the review of reference lists in systematic reviews of the area.A total of 185 studies were discarded due to duplication.Then, the remaining 230 articles were thoroughly reviewed.As a result, 26 articles met all the inclusion criteria (Figure 1).
Eight of the studies in our systematic review specifically investigated the association between TNF-alpha gene polymorphisms and severe dengue in Latin America.These studies focused on various single nucleotide polymorphisms (SNPs) within the TNF-alpha gene, including -308G>A, -238G>A, -857C>T, and -1031T>C.The results of these studies varied: while some reported significant associations between specific TNF-alpha SNPs and severe dengue, others found no significant correlations between these two factors.This heterogeneity may have resulted from differences in study populations, sample sizes, and genotyping techniques.

Quality of the studies
In total, 46.2% of the 26 articles (n=12) presented some concern or risk of bias in their selection of cases and controls.The selection of cases was mainly based on WHO criteria from 1997 or 2009, with clinical confirmation through RT-PCR tests.In their assessment of comparability between groups, 34.6% of studies had a high risk of bias.Lastly, 96.2% of the studies presented some concern or a high risk of bias in their exposure assessment-they did not present any information on losing participants.Supplementary Table S4 presents the eight quality review criteria for each study included in this review.

DISCUSSION
This systematic review demonstrated the existence of multiple polymorphisms in immune system genes that are related to the clinical outcomes of dengue virus infections.As Table 1 summarizes, 17 of the 26 articles reported finding polymorphisms that are associated with the risk of severe dengue (Risk) and 17 articles reported detecting polymorphisms that are associated with a decreased risk of severe dengue (Protective).Notably, most of these studies were conducted in Brazil (n=12).
Regarding the immune response in dengue virus infection, both innate and adaptive responses play an essential role in defending organisms infected with severe dengue, and that the regulation of these responses directly impacts the clinical outcome of the disease 58 .One of the first mechanisms employed by the innate immune system is the production of interferon and proinflammatory cytokines to trigger the initial response against the virus through dendritic cells 59 .However, this mechanism may lead to cell permeability and fluid leakage.
Most genes reported in the analyzed studies are related to the innate immune response.Polymorphisms in Toll-like    receptors responsible for recognizing viral proteins 48 ; genes associated with the production of interleukins capable of inhibiting the synthesis of proinflammatory cytokines and suppressing the ability of cells to present antige ns 33,34,38,39,43,44,49,50,55,57 ; and genes coding for surface proteins in multiple cells of the immune system, such as the type C lecithin receptor (DC-SIGN) 39,46,47 , MBL2 35,36 (it should be noted that Ornelas et al. 36 only observed such an association after haplotype analyses), and CLEC5A 39,40,46,47 .One study also reported a protective effect mediated by complement 37 .
The substantial number of studies focused on TNF-alpha gene polymorphisms in this review highlights the importance of this cytokine in severe dengue pathogeneses.The inconsistent findings among these studies call attention to the complexity of the role of TNF-alpha in individuals' susceptibility to severe dengue and the need for further investigation.Larger, multi-center studies with standardized methodologies and thorough genetic analysis are needed to clarify the association between TNF-alpha gene polymorphisms and severe dengue risks.Future research should also explore the role of interactions between TNF-alpha polymorphisms and other immune system genes in the pathogenesis of dengue, to further elucidate the genetic factors influencing individuals' susceptibility to severe dengue.
Concerning cellular immunity, the activation of CD4+ and CD8+ T lymphocytes is essential for eliminating infected cells.However, T cells may cause immunopathology during DENV infections, in a phenomenon called original antigenic sin, in which the activation of memory lymphocytes generates an elevated production of proinflammatory cytokines with the consequences described above 60 .Studies by Falcón et al. 45 , Fernández-Mestre et al. 56 , García et al. 53 , and Sierra et al. 54 documented the influence of polymorphisms in the major histocompatibility complex responsible for the regulation of the immune system through the process of antigen presentation.García et al. 52 in 2010 and Noecker et al. 46 in 2014 also investigated this immune system and identified polymorphisms in the FcγRIIa gene, which encodes cell surface proteins that mediate responses in B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, among others.Other studies in our review focused on verifying the relationship between genetic immunity and the single nucleotide polymorphism (SNP) of G2431A IDO1, OSBPL10, and HLA-DRB1 19,41,42,51 .
We noted some similarities and differences between the studies we analyzed and other studies concerning innate immunity polymorphisms: some reports from Thailand indicated that rs4804803 (CD209) and rs3753394 (CFH) polymorphisms were not linked to dengue 25 .Similarly, the current review found no significant association between this polymorphism and severe disease in Latin American countries 38,47 .However, another study conducted with the Thai population confirmed CD209 with an OR=5.84 (2.77-12.31) of DHF compared to DF 25 .
While analyzing this same component of innate immunity, a study conducted in India confirmed OR=0.39 (0.16-0.88) of severe disease associated with rs3775291 (TLR3) 25 .In contrast, no association between these factors was found in the Latin American population 48 .Lastly, contrary to what was found in Indonesia regarding the TLR4 gene (no significant association with disease) 25 , our study confirmed a protective effect of TLR4-rs2737190-G/G/G in cases of severe dengue fever 48 .
While analyzing the genetic immunity groups, our study found significant associations of the MICB gene with the risk of severe dengue 53 .Previous studies conducted with the Asian population observed a similar scenario, confirming that the same increase in risk affected DSS: 1.58 (1.02-2.40)odds of DSS compared to non-DSS 25 .Differences between countries in Latin America and those in other continents may occur due to specific ethnicity factors resulting in cases in which Asians are protected by polymorphisms but Latin Americans are not, or on the contrary, for some polymorphisms the protective effect occurs in Latin Americans but not in Asians.
Dengue is currently a public health problem in most Latin American countries.Since 2009, the disease has expanded its distribution, causing periodic epidemics with a constant raise in cases.Identifying the polymorphisms that affect dengue can help researchers find early markers that make it easier to predict the clinical outcome of this disease and may even be helpful in designing vaccines.Genetic studies are essential for gathering information on circulating viruses and creating a better understanding of DENV transmission and epidemiology in a specific region.

CONCLUSION
Lastly, it is crucial to consider the time span of the studies included in this review, where the most recent studies occurred in 2020 33,44,48 , and some were conducted almost two decades ago.The different biases previously described for each of the studies resulted in a high subjective heterogeneity.On the other hand, the lack of studies in most Latin American countries, probably due to low research funding, has hampered a thorough investigation of the potential of markers in preventing severe dengue in this region.
Nevertheless, this review provides an overview of the genetic aspects associated with severe dengue in this region, which is an essential analysis, considering the diversity that could be expected in this regard and the fact that these types of studies are mainly published in Asian countries.Despite the aforementioned scenario, the efforts made to understand the clinical course of patients with dengue are fully justified.The evidence collected so far will hopefully serve as a basis for improving disease prediction methods, positively affecting the early identification of cases that require greater health care attention.

Figure 1 -
Figure 1 -PRISMA flowchart of the strategy used to identify papers assessing polymorphism genetics and severe dengue

Figure 2 -
Figure 2 -Summary of the assessment of risks of bias in the included articles.The classification is based on the Newcastle-Ottawa Quality Assessment Scale for Case-Control Studies (NOS).

Table 1 -
Characteristics of the included papers assessing polymorphism genetics and severe dengue.

Table 1 -
Characteristics of the included papers assessing polymorphism genetics and severe dengue.(cont.)

Table 1 -
Characteristics of the included papers assessing polymorphism genetics and severe dengue.(cont.)

Table 1 -
Characteristics of the included papers assessing polymorphism genetics and severe dengue.(cont.)

Table 1 -
Characteristics of the included papers assessing polymorphism genetics and severe dengue.(cont.)